In vitro Determination of Rapamycin-triggered FKBP-FRB Interactions Using a Molecular Tension Probe

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Protein-protein interactions (PPI) are crucial in biological systems but challenging to study in vitro.
• Existing imaging schemes for PPI are often limited to cellular or in vivo environments.
• The in vitro validation of PPI-based bioanalytical imaging is not well-established.

Purpose of the Study:

• To demonstrate and characterize in vitro protein-protein interactions (PPI) within a purified single-chain bioluminescent probe.
• To investigate the influence of a ligand on the molecular kinetics of the probe.
• To identify optimal substrates for enhanced bioluminescence detection.

Main Methods:

• Construction and purification of a single-chain probe (TP2.4) containing an artificial luciferase (ALuc) flanked by FKBP and FRB domains.
• In vitro kinetic analysis of the probe's interaction with its ligand and substrates.
• Circular dichroism (CD) spectroscopy to assess conformational changes.

Main Results:

• The TP2.4 probe successfully demonstrated in vitro PPI upon ligand recognition.
• Rapamycin significantly enhanced the enzymatic affinities (Km) of TP2.4 to its substrates, with minimal impact on turnover rates (Kcat) and maximal velocity (Vmax).
• Circular dichroism indicated a minor increase in alpha-helical content upon rapamycin binding. A coelenterazine derivative, 6-N3-CTZ, showed the highest catalytic efficiency and photon count variance.

Conclusions:

• This study provides the first in vitro demonstration of intramolecular PPI in a purified single-chain bioluminescent probe.
• Ligand binding modulates the kinetic properties of the probe, primarily affecting substrate affinity.
• The findings offer insights into the biochemical factors influencing PPI in vitro and highlight the potential of such probes for bioanalytical applications.